Methods and compounds for polynucleotide synthesis

ABSTRACT

Methods of forming polynucleotides are disclosed.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The U.S. government may have a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of MDA CONTRACTN39998-01-9-7068 awarded by the DARPA of the U.S. Government.

BACKGROUND

Much interest has been focused on reactions for coupling nucleotides toform polynucleotide chains, and various chemical schemes have beendescribed for the synthesis of polynucleotides. Typically these methodsuse a nucleoside reagent of the formula:

in which:

A represents H, OH, halogen, or an optionally protected hydroxyl group;

B is a purine or pyrimidine base whose exocyclic amine functional groupis optionally protected;

one of M or Q is a conventional protective group for the 3′ or 5′—OHfunctional group while the other is:

where x may be 0 or 1, provided that:

a) when x=1:

R′ represents H and R″ represents a negatively charged oxygen atom; or

R′ is an oxygen atom and R″ represents either an oxygen atom or anoxygen atom carrying a protecting group; and

b) when x=0, R′ is an oxygen atom carrying a protecting group and R″ iseither a hydrogen or a di-substituted amine group.

When x is equal to 1, R′ is an oxygen atom and R″ is an oxygen atom, themethod is in this case the so-called phosphodiester method; when R″ isan oxygen atom carrying a protecting group, the method is in this casethe so-called phosphotriester method.

When x is equal to 1, R′ is a hydrogen atom and R″ is a negativelycharged oxygen atom, the method is known as the H-phosphonate method.

When x is equal to 0, R′ is an oxygen atom carrying a protecting groupand R″ is a halogen, the method is known as the phosphite method, andwhen R″ is a leaving group of the disubstituted amine type, the methodis known as the phosphoramidite method.

The conventional sequence used to prepare an oligonucleotide usingreagents of the type of formula (I), basically follows four separatesteps: (a) coupling a selected nucleoside which also has a protectedhydroxy group, through a phosphite linkage to a functionalized supportin the first iteration, or a nucleoside bound to the substrate (i.e.,the nucleoside-modified substrate) in subsequent iterations; (b)optionally, but preferably, blocking unreacted hydroxyl groups on thesubstrate bound nucleoside; (c) oxidizing the phosphite linkage of step(a) to form a phosphate linkage; and (d) removing the protecting group(“deprotection”) from the now substrate-bound nucleoside coupled in step(a), to generate a reactive site for the next cycle of these steps. Thefunctionalized support (in the first cycle) or deprotected couplednucleoside (in subsequent cycles) provides a substrate-bound moiety witha linking group for forming the phosphite linkage with a next nucleosideto be coupled in step (a). Final deprotection of nucleoside bases can beaccomplished using alkaline conditions such as ammonium hydroxide,methyl amine, ethanolamine, or others, in a known manner.

The foregoing methods of preparing polynucleotides are well known anddescribed in detail, for example, in Caruthers, Science 230: 281-285,1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar etal., Nature 310: 105-110, 1984; and in “Synthesis of OligonucleotideDerivatives in Design and Targeted Reaction of OligonucleotideDerivatives”, CRC Press, Boca Raton, Fla. pages 100 et seq., U.S. Pat.No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S.Pat. No. 5,153,319, U.S. Pat. No. 5,869,643, EP 0294196, and elsewhere.The phosphoramidite and phosphite triester approaches are most broadlyused, but other approaches include the phosphodiester approach, thephosphotriester approach and the H-phosphonate approach. Such approachesare described in Beaucage et al., Tetrahedron (1992) 12:2223-2311. Amore recent approach for synthesis of polynucleotides is described inU.S. Pat. No. 6,222,030 B1 to Dellinger et al, Issued Apr. 24, 2001.

In the typical phosphoramidite method of solid phase oligonucleotidesynthesis, the synthesis typically proceeds in the 3′ to 5′ direction(referring to the sugar component of the added nucleoside), although thesynthesis may easily be conducted in the reverse direction. The addednucleoside generally has a dimethoxytrityl protecting group on its 5′hydroxyl and a phosphoramidite functionality on its 3′ hydroxylposition. Beaucage et al. (1981) Tetrahedron Lett. 22:1859. See FIG. 1for a schematic representation of this technology. In FIG. 1 “B”represents a purine or pyrimidine base, “DMT” represents dimethoxytritylprotecting group and “iPr” represents isopropyl. In the first step ofthe synthesis cycle, the “coupling” step, the 5′ end of the growingchain is coupled with the 3′ phosphoramidite of the incoming monomer toform a phosphite triester intermediate (the 5′ hydroxyl protecting groupprevents more than one monomer per synthesis cycle from attaching to thegrowing chain). Matteucci et al. (1981) J. Am. Chem. Soc. 103:3185.Next, the optional “capping reaction” is used to stop the synthesis onany chains having an unreacted 5′ hydroxyl, which would be onenucleotide short at the end of synthesis. The “capping reaction” has theadded benefit of cleaving coupling products from the heterobases, suchas O-6 adducts on guanosine, prior to the oxidation step. The phosphitetriester intermediate is subjected to oxidation (the “oxidation” step)after each coupling reaction to yield a more stable phosphotriesterintermediate. Without oxidation, the unstable phosphite triester linkagewould cleave under the acidic conditions of subsequent synthesis steps.Letsinger et al. (1976) J. Am. Chem. Soc. 98:3655. Removal of the 5′protecting group of the newly added monomer (the “deprotection” step) istypically accomplished by reaction with acidic solution to yield a free5′ hydroxyl group, which can be coupled to the next protected nucleosidephosphoramidite. This process is repeated for each monomer added untilthe desired sequence is synthesized.

According to some protocols, the synthesis cycle of couple, cap,oxidize, and deprotect is shortened by omitting the capping step or bytaking the oxidation step ‘outside’ of the cycle and performing a singleoxidation reaction on the completed chain. For example, oligonucleotidesynthesis according to H-phosphonate protocols will permit a singleoxidation step at the conclusion of the synthesis cycles. However,coupling yields are less efficient than those for phosphoramiditechemistry and oxidation requires longer times and harsher reagents thanamidite chemistry.

Conventional synthesis protocols of oligonucleotides are not withoutdisadvantages. For example, cleavage of the DMT protecting group underacidic conditions gives rise to the resonance-stabilized and long-livedbis(p-anisyl)phenylmethyl carbocation. Gilham et al. (1959) J. Am. Chem.Soc. 81:4647. Protection and deprotection of hydroxyl groups with DMTare thus readily reversible reactions, resulting in incomplete reactionsduring oligonucleotide synthesis giving rise to sequence deletions and alower yield than might otherwise be obtained. To circumvent suchproblems, large excesses of acid are used with DMT to achievequantitative deprotection. However, the repeated exposure of DNAsequences to acids gives rise to acid catalyzed removal of theheterobases from the sugar ring. Heterobase removal is most facile onthe purine bases, adenosine, and guanosine; resulting in depurination,however all heterobases and modified heterobases can be susceptible toacid catalyzed removal. As bed volume of the polymer is increased inlarger scale synthesis, increasingly greater quantities of acid arerequired. The acid-catalyzed depurination that occurs during thesynthesis of oligodeoxyribonucleotides is thus increased by the scale ofsynthesis. Caruthers et al., in Genetic Engineering: Principles andMethods, J. K. Setlow et al., Eds. (New York: Plenum Press, 1982).Solvent use in larger scale synthesis becomes increasingly prohibitiveas well, as more washing is required. In particular, the reagents usedin the coupling step typically are highly susceptible to hydrolysis,which requires dry solvents, further increasing the cost of solvents.

SUMMARY

Briefly described, embodiments of this disclosure include method offorming polynucleotides. One exemplary method, among others, includesproviding a structure X selected from structure C′ and O′ illustratedherein; where R1 is selected from substituted carbonyls, substitutedsilanes and functional groups, wherein the functional group is selectedfrom carbonate, ester, amide, carbamate, silane, siloxane, orthoester,acetal, and ketal; R2 is selected from benzyl, substituted benzyl,phenyl, substituted phenol, tertiary alkyl, substituted tertiary alkyl;B comprises purine and pyrimidine bases and analogs thereof; wherein nis from 1 to 350; and wherein ● is a substrate; and oxidizing anddeprotecting structure X simultaneously, wherein the aqueous buffersolution is an oxidant and a nucleophile at a pH of about 6 to 10.

Additional objects, advantages, and novel features of this disclosureshall be set forth in part in the descriptions and examples that followand in part will become apparent to those skilled in the art uponexamination of the following specifications or may be learned by thepractice of the disclosure. The objects and advantages of the disclosuremay be realized and attained by means of the instruments, combinations,compositions, and methods particularly pointed out in the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference is now made to the following drawings. Note that thecomponents in the drawings are not necessarily to scale.

FIG. 1 schematically illustrates a prior art multi-steppolynucleotide/oligonucleotide synthesis method.

FIG. 2 schematically illustrates an embodiment of the two-steppolynucleotide/oligonucleotide synthesis method in the 5′ to 3′direction.

FIG. 3 illustrates embodiments of structures produced in the synthesisdescribed in FIG. 2.

FIG. 4 schematically illustrates another embodiment of the two-steppolynucleotide/oligonucleotide synthesis method in the 3′ to 5′direction.

FIG. 5 illustrates embodiments of structures produced in the synthesisdescribed in FIG. 2.

DETAILED DESCRIPTION

Embodiments of the present disclosure will employ, unless otherwiseindicated, conventional techniques of synthetic organic chemistry,biochemistry, molecular biology, and the like, which are within theskill of one in the art. Such techniques are explained fully in theliterature.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions disclosed and claimedherein. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

Before the embodiments of the present disclosure are described indetail, it is to be understood that unless otherwise indicated thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such mayvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps may be executed in different sequence where this is logicallypossible.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meanings,unless a contrary intention is apparent.

As used herein, polynucleotides include single or multiple strandedconfigurations, where one or more of the strands may or may not becompletely aligned with another. The terms “polynucleotide” and“oligonucleotide” shall be generic to polydeoxynucleotides (containing2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to anyother type of polynucleotide which is an N-glycoside of a purine orpyrimidine base, and to other polymers in which the conventionalbackbone has been replaced with a non-naturally occurring or syntheticbackbone or in which one or more of the conventional bases has beenreplaced with a non-naturally occurring or synthetic base.

A “nucleotide” refers to a sub-unit of a nucleic acid (whether DNA orRNA or analogue thereof) which includes a phosphate group, a sugar groupand a nitrogen containing base, as well as analogs of such sub-units.

A “nucleoside” references a nucleic acid subunit including a sugar groupand a nitrogen containing base. It should be noted that the term“nucleotide” is used herein to describe embodiments of the disclosure,but that one skilled in the art would understand that the term“nucleoside” and “nucleotide” are interchangable in most instances. Oneskilled in the art would have the understanding that additionalmodification to the nucleoside may be necessary and one skilled in theart has such knowledge.

A “nucleoside moiety” refers to a molecule having a sugar group and anitrogen containing base (as in a nucleoside) as a portion of a largermolecule, such as in a polynucleotide, oligonucleotide, or nucleosidephosphoramidite.

A “nucleotide monomer” refers to a molecule which is not incorporated ina larger oligo- or poly-nucleotide chain and which corresponds to asingle nucleotide sub-unit; nucleotide monomers may also have activatingor protecting groups, if such groups are necessary for the intended useof the nucleotide monomer.

A “polynucleotide intermediate” references a molecule occurring betweensteps in chemical synthesis of a polynucleotide, where thepolynucleotide intermediate is subjected to further reactions to get theintended final product (e.g., a phosphite intermediate, which isoxidized to a phosphate in a later step in the synthesis), or aprotected polynucleotide, which is then deprotected.

An “oligonucleotide” generally refers to a nucleotide multimer of about2 to 100 nucleotides in length, while a “polynucleotide” includes anucleotide multimer having any number of nucleotides greater than 1.

It will be appreciated that, as used herein, the terms “nucleoside” and“nucleotide” will include those moieties which contain not only thenaturally occurring purine and pyrimidine bases, e.g., adenine (A),thymine (T), cytosine (C), guanine (G), or uracil (U), but also modifiedpurine and pyrimidine bases and other heterocyclic bases which have beenmodified (these moieties are sometimes referred to herein, collectively,as “purine and pyrimidine bases and analogs thereof”). Suchmodifications include, e.g., diaminopurine and its deravitives, inosineand its deravitives, alkylated purines or pyrimidines, acylated purinesor pyrimidines thiolated purines or pyrimidines, and the like, or theaddition of a protecting group such as acetyl, difluoroacetyl,trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl,phenoxyacetyl, dimethylformamidine, N,N-diphenyl carbamate, or the like.The purine or pyrimidine base may also be an analog of the foregoing;suitable analogs will be known to those skilled in the art and aredescribed in the pertinent texts and literature. Common analogs include,but are not limited to, 1-methyladenine, 2-methyladenine,N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine,N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine,5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine,2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil,5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil,5-(carboxymethylaminomethyl)-uracil, 2-thiouracil,5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, pseudouracil,1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine,xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine and2,6-diaminopurine.

An “internucleotide bond” refers to a chemical linkage between twonucleoside moieties, such as a phosphodiester linkage in nucleic acidsfound in nature, or such as linkages well known from the art ofsynthesis of nucleic acids and nucleic acid analogues. Aninternucleotide bond may include a phospho or phosphite group, and mayinclude linkages where one or more oxygen atoms of the phospho orphosphite group are either modified with a substituent or replaced withanother atom, e.g., a sulfur atom, or the nitrogen atom of a mono- ordi-alkyl amino group.

An “array”, unless a contrary intention appears, includes any one, two,or three dimensional arrangement of addressable regions bearing aparticular chemical moiety or moieties (for example, polynucleotidesequences) associated with that region. An array is “addressable” inthat it has multiple regions of different moieties (for example,different polynucleotide sequences) such that a region (a “feature” or“spot” of the array) at a particular predetermined location (an“address”) on the array will detect a particular target or class oftargets (although a feature may incidentally detect non-targets of thatfeature). In the case of an array, the “target” will be referenced as amoiety in a mobile phase (typically fluid), to be detected by probes(“target probes”) which are bound to the substrate at the variousregions. However, either of the “target” or “target probes” may be theone which is to be evaluated by the other (thus, either one could be anunknown mixture of polynucleotides to be evaluated by binding with theother). While probes and targets of the present disclosure willtypically be single-stranded, this is not essential. An “array layout”refers to one or more characteristics of the array, such as featurepositioning, feature size, and some indication of a moiety at a givenlocation. “Hybridizing” and “binding”, with respect to polynucleotides,are used interchangeably.

A “group” includes both substituted and unsubstituted forms. Typicalsubstituents include one or more lower alkyl, any halogen, hydroxy, oraryl, or optionally substituted on one or more available carbon atomswith a nonhydrocarbyl substituent such as cyano, nitro, halogen,hydroxyl, or the like. Any substituents are typically chosen so as notto substantially adversely affect reaction yield (for example, not lowerit by more than 20% (or 10%, or 5% or 1%) of the yield otherwiseobtained without a particular substituent or substituent combination).An “acetic acid” includes substituted acetic acids such asdi-chloroacetic acid (DCA) or tri-chloroacetic acid (TCA).

A “phospho” group includes a phosphodiester, phosphotriester, andH-phosphonate groups. In the case of either a phospho or phosphitegroup, a chemical moiety other than a substituted 5-membered furyl ringmay be attached to O of the phospho or phosphite group which linksbetween the furyl ring and the P atom.

A “protecting group” is used in the conventional chemical sense toreference a group, which reversibly renders unreactive a functionalgroup under specified conditions of a desired reaction. After thedesired reaction, protecting groups may be removed to deprotect theprotected functional group. All protecting groups should be removable(and hence, labile) under conditions which do not degrade a substantialproportion of the molecules being synthesized. In contrast to aprotecting group, a “capping group” permanently binds to a segment of amolecule to prevent any further chemical transformation of that segment.

A “hydroxyl protecting group” refers to a protecting group where theprotected group is a hydroxyl. A “reactive-site hydroxyl” is theterminal 5′-hydroxyl during 3′-5′ polynucleotide synthesis and is the3′-hydroxyl during 5′-3′ polynucleotide synthesis. An “acid labileprotected hydroxyl” is a hydroxyl group protected by a protecting groupthat can be removed by acidic conditions. Similarly, an “acid labileprotecting group” is a protecting group that can be removed by acidicconditions. Preferred protecting groups that are capable of removalunder acidic conditions (“acid-labile protecting groups”) include thosesuch as tetrahydropyranyl groups, e.g., tetrahydropyran-2-yl and4-methoxytetrahydropyran-2-yl; an arylmethyl group with n aryl groups(where n=1 to 3) and 3-n alkyl groups such as an optionally substitutedtrityl group, for example a monomethoxytrityl for oligoribonucleotidesynthesis and a dimethoxytrityl for oligodeoxyribonucleotide synthesis;pixyl; isobutyloxycarbonyl; t-butyl; and dimethylsilyl. A trityl groupis a triphenylmethyl group. Suitable protecting groups are described in“Protective Groups in Organic Synthesis” by T.W. Green, WileyInterscience.

The term “alkyl” as used herein, unless otherwise specified, refers to asaturated straight chain, branched or cyclic hydrocarbon group of 1 to24, typically 1-12, carbon atoms, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl,neopentyl, hexyl, isohexyl, cyclohexyl, 3-methylpentyl,2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term “lower alkyl” intendsan alkyl group of one to eight carbon atoms, and includes, for example,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl,cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term“cycloalkyl” refers to cyclic alkyl groups such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

The term “aryl” as used herein refers to an aromatic species containing1 to 5 aromatic rings, either fused or linked, and either unsubstitutedor substituted with 1 or more substituents typically selected from thegroup consisting of amino, halogen, and lower alkyl. Preferred arylsubstituents contain 1 to 3 fused aromatic rings, and particularlypreferred aryl substituents contain 1 aromatic ring or 2 fused aromaticrings. Aromatic groups herein may or may not be heterocyclic. The term“aralkyl” intends a moiety containing both alkyl and aryl species,typically containing less than about 24 carbon atoms, and more typicallyless than about 12 carbon atoms in the alkyl segment of the moiety, andtypically containing 1 to 5 aromatic rings. The term “aralkyl” willusually be used to refer to aryl-substituted alkyl groups. The term“aralkylene” will be used in a similar manner to refer to moietiescontaining both alkylene and aryl species, typically containing lessthan about 24 carbon atoms in the alkylene portion and 1 to 5 aromaticrings in the aryl portion, and typically aryl-substituted alkylene.Exemplary aralkyl groups have the structure —(CH₂)_(j)—Ar wherein j isan integer in the range of 1 to 24, more typically 1 to 6, and Ar is amonocyclic aryl moiety.

Discussion

Embodiments of the present disclosure include methods for fabricatingoligonucleotides and polynucleotides (herein after “polynucleotide”),methods of deprotecting the hydroxyl on the sugar moiety of a nucleotideand deprotecting the nascent internucleotide bond simultaneously,protecting groups for the hydroxyl group on the sugar moiety of anucleotide, protecting groups for the nascent internucleotide bondbetween two nucleotides, and nucleotide compounds including protectinggroups for the hydroxyl group on the sugar moiety of a nucleotide andprotecting groups for the nascent internucleotide bond between twonucleotides, all of which have numerous advantages relative to priormethods such as those discussed above.

In general, the methods involve forming a target biopolymer (hereinafter“target polynucleotide”) using nucleotide compounds including protectinggroups for the hydroxyl group on the sugar moiety of a nucleotide andprotecting groups for the nascent internucleotide bond between twonucleotides. In general, the methods involve a coupling step and adeprotection and oxidative step, where the deprotection and oxidativesteps occur simultaneously. In the simultaneous deprotection andoxidative step, deprotecting the hydroxyl on the sugar moiety of thenucleotide and deprotecting the nascent internucleotide bond between twoadjacent nucleotides occurs simultaneously or substantiallysimultaneously. In particular, the simultaneous deprotection andoxidative steps are substantially irreversible or irreversible.Substantially irreversible means at least 80% irreversible, at least 90%irreversible, and at least 95% irreversible.

In addition, the number of steps used in the methods for synthesizingthe target polynucleotide is reduced relative to that shown in FIG. 1.In particular, the methods involve a two-step synthesis cycle that canbe used to produce target polynucleotides in either the 5′ to 3′direction or the 3′ to 5′ direction. As mentioned above, the methodsinvolve a coupling step and a deprotection and oxidative step that canbe repeated in an iterative manner to produce the target polynucleotideof interest.

FIG. 2 schematically illustrates an embodiment of the two-step targetpolynucleotide synthesis method in the 5′ to 3′ direction under typicalconditions. Structure A is provided and coupled with Structure B to formStructure C. The “B” moiety includes purine and pyrimidine bases andanalogs thereof. “R1” can include groups such as, but not limited to,substituted carbonyls, substituted silanes and functional groups suchas, carbonate, ester, amide, carbamate, silane, siloxane, orthoester,acetal, ketal, and the like. In particular, “R1” can include groups suchas, but not limited to, oxycarbonate, aryl ester, alkyl ester, alkylsilane, aryl silane, alkylsiloxane, arylsiloxane, alkylarylsilane, andalkylarylsiloxane. Further, “R1” can include, but is not limited to,aryloxycarbonyl (Arco), methylthiophenyloxycarbonyl,flurormethylthiophenyloxycarbonyl, methoxymethylthiobenzylcarbonyl,dimethylthiophenylcarbonyl, acetylsalicylate, diacetylphenoxysalicylate,methylthioacetylsalicylate, dimethylthioacetylsalicylate,nitroacetylsalicylate, fluoroacetylsalicylate,cyclododecyloxydi(trimethoxysilyl)siloxane,diphenylmethoxydi(trimethoxysilyl)siloxane, and the like. “R2” caninclude groups such as, but not limited to, benzyl, substituted benzyl,phenyl, substituted phenol, tertiary alkyl, substituted tertiary alkyl,and the like. In particular, “R2” can include groups such as, but notlimited to, alkylbenzyl, dialkylbenzyl, trialkylbenzyl, thioalkylbenzyl,phenylthiobenzyl, dithioalkylbenzyl, trithioalkylbenzyl,thioalkylhalobenzyl, alkyloxybenzyl, dialkyloxybenzyl, halobenzyl,dihalobenzyl, trihalobenzyl, esterifiedsalicyl, and the like. Further,“R2” can include, but is not limited to, 2-methoxybenzyl,methylthiophenyloxycarbonyl, flurormethylthiophenyloxycarbonyl,methoxymethylthiobenzylcarbonyl, dimethylthiophenylcarbonyl,acetylsalicyl, diacetylphenoxysalicyl, methylthioacetylsalicyl,dimethylthioacetylsalicyl, nitroacetylsalicyl, fluoroacetylsalicyl, andthe like.

Structure C is oxidized and deprotected simultaneously or substantiallysimultaneously in an aqueous buffer solution to form Structure D. Theaqueous buffer solution includes a compound or compound mixture thatacts as both an oxidant and a nucleophile at a pH of about 6 to 10,about 6.5 to 8.5, about 6.5 to 8, and about 7 to 8. This mixture caninclude oxidizing agents that act as nucleophiles such as, but notlimited to, hydroperoxides or peracids or mixtures of oxidizing agentthat do and do not act as nucleophiles such as, but not limited to,mixtures of peroxides and peracids. The aqueous buffer solution includescompounds that have a pKa from about 1 to 11, about 3 to 11, about 7 to11. The aqueous buffer solution includes compounds such as, but notlimited to, hydrogen peroxide; peracids; performic acid; peracetic acid;perbenzoic acid; chloroperbenzoic acid, and the like; hydroperoxides;butylhydroperoxide; benzylhydroperoxide; phenylhydroperoxide; othersimilar compounds; and combinations thereof.

In addition, the aqueous buffer solution includes a buffer such as, butnot limited to, tris(hydroxymethyl)aminomethane, aminomethylpropanol,citric acid, N,N′-bis(2-hydroxyethyl)glycine,2-[bis(2-hydroxyethyl)amino]-2-(hydroxy-methyl)-1,3-propanediol,2-(cyclohexylamino)ethane-2-sulfonic acid,N-2-hydroxyethyl)piperazine-N′-2-ethane sulfonic acid,N-(2-hydroxyethyl)piperazine-N′-3-propane sulfonic acid,morpholinoethane sulfonic acid, morpholinopropane sulfonic acid,piperazine-N,N′-bis(2-ethane sulfonic acid),N-tris(hydroxymethyl)methyl-3-aminopropane sulfonic acid,N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid,N-tris(hydroxymethyl)methylglycine, and combinations thereof.

The hydrogen peroxide, peracids, and hydroperoxides are typically lessthan 30% weight/vol, more typically between 0.1% and 10% weight/vol andmost typically 1% to 5% weight/vol.

The deprotection and oxidation occur substantially simultaneously orsimultaneously, in other words both reactions are occurring at the sametime. The rate of the two reactions can be different and therefore thecompetition times can be different. In the case of certain reactions,oxidation of a protecting group can result in enhanced rates ofdeprotection. It is most desirable if the deprotection reaction issubstantially irreversible or irreversible. Although not intending to bebound by theory, the substantially irreversible nature of the reactionis due, at least in part, because the “R1” group breaks apart into threecomplexes, which substantially decrease the chance of a reverse reactionto reform “R1”. Irreversibility is a desireable property of most if notall protecting groups and is equally desirable for R2.

The two-step process can be repeated in an iterative manner by replacingStructure B with the reaction product produced after each deprotectionand oxidation step. For example, Structure D is coupled with StructureA. FIG. 3 illustrates embodiments of Structure C′ and Structure D′,which represent structures that could be produced in an iterative mannerusing the method described in FIG. 2. “n” can be 1 to 350, 5 to 150, and15 to 100, and depends on the length of the target polynucleotide to besynthesized.

The chemical synthesis method described herein is advantageous, in thatthe growing nucleic acid chain is exposed to fewer reagents and thesynthesis cycle can be performed using simpler automation than withstandard processes. When growing oligonucleotides are exposed to fewerchemical reagents, there are fewer chances of undesired chemicalmodifications, and those chemical modifications can be more easilycontrolled. As a manufacturing issue, simple automation can result in amore robust and reproducible manufacturing process. The non-reversiblenature of the reactions is especially advantageous for synthesis onmicroscale and on planar surfaces. With highly reversible reactions itcan be very difficult to achieve reaction completion under theseconditions. The reactions are typically carried out under more stringentconditions to completely remove the reagent byproducts from the surface.

FIG. 4 schematically illustrates an embodiment of the two-step targetpolynucleotide synthesis method in the 3′ to 5′ direction under typicalconditions. Structure M is provided and coupled with Structure N to formStructure O. “B”, R1, and R2 are the same as those described inreference to FIG. 2 and the corresponding text.

Structure O is oxidized and deprotected simultaneously or substantiallysimultaneously in an aqueous buffer solution to form Structure P. Theaqueous buffer solution is the same as that described in reference toFIG. 2 and the corresponding text.

In addition, the aqueous buffer solution includes a buffer as describedabove.

The two-step process can be repeated in an iterative manner by replacingStructure N with the reaction product produced after each deprotectionand oxidation step. For example, Structure P is coupled with StructureM. FIG. 5 illustrates embodiments of Structure O′ and Structure P′,which represent structures that could be produced in an iterative mannerusing the method described in FIG. 4. “n” can be 1 to 350, 5 to 150, and15 to 100, and depends on the length of the target polynucleotide to besynthesized.

As mentioned above, embodiments of the methods lend themselves tosynthesis of polynucleotides on array substrates in either the 3′-to-5′or the 5′-to-3′ direction. In the former case, the initial step of thesynthetic process involves attachment of an initial nucleoside to thearray substrate at the 3′ position, leaving the 5′ position availablefor covalent binding of a subsequent monomer. In the latter case, theinitial step of the synthetic process involves attachment of an initialnucleoside to the array substrate at the 5′ position, leaving the 3′position available for covalent binding of a subsequent monomer.Following synthesis, the polynucleotide may, if desired, be cleaved fromthe solid support. The details of the synthesis in either the 3′-to-5′or the 5′-to-3′ direction will be readily apparent to the practitionerof ordinary skill, based on the prior art and the disclosure containedherein.

In addition, embodiments of the present disclosure provide methods ofgenerating addressable arrays of polynucleotides on a substrate.Embodiments of this method include a solution having at least one of anucleoside, a nucleotide, an oligonucleotide, or a polynucleotide, thatis contacted with an array substrate to form a an oligonucleotide or apolynucleotide, and in an iterative manner, the target polynucleotide ofinterest can be fabricated according to and using one or more methodsand nucleotide compounds as described herein.

In one such embodiment, at each of the multiple different addresses onthe substrate (e.g., at least one hundred, at least one thousand, or atleast ten thousand addresses), the in situ synthesis cycle is repeatedso as to form the addressable array with the same or differentpolynucleotide sequences at one or more different addresses on thesubstrate. In the array forming method, the compounds to be coupled atrespective addresses are deposited as droplets at those addresses using,for example, an inkjet printing system. The polynucleotides can beproduced by disposing solutions (e.g., selected from four solutions,each containing a different nucleotide) on particular addressablepositions in a specific order in an iterative process according themethods described herein.

The disclosure also encompasses the formation of an internucleotide bondbetween two polynucleotides or oligonucleotides, or between apolynucleotide and an oligonucleotide, resulting in an extendedpolynucleotide immobilized on the array surface. In such case, one ofthe polynucleotides or oligonucleotides is dissolved in a solvent andtarget polynucleotides can be made according to the methods describedherein.

The array may contain any number of features, generally including atleast tens of features, usually at least hundreds, more usuallythousands, and as many as a hundred thousand or more features. All ofthe features may be different, or some or all could be the same. Eachfeature carries a predetermined moiety or a predetermined mixture ofmoieties, such as a particular polynucleotide sequence or apredetermined mixture of polynucleotides. The features of the array maybe arranged in any desired pattern (e.g., organized rows and columns offeatures, for example, a grid of features across the substrate surface);a series of curvilinear rows across the substrate surface (for example,a series of concentric circles or semi-circles of features), and thelike. In embodiments where very small feature sizes are desired, thedensity of features on the substrate may range from at least about tenfeatures per square centimeter, or at least about 35 features per squarecentimeter, or at least about 100 features per square centimeter, and upto about 1000 features per square centimeter, up to about 10,000features per square centimeter, or up to 100,000 features per squarecentimeter. Each feature carries a predetermined nucleotide sequence(which includes the possibility of mixtures of nucleotide sequences).

In one embodiment, about 10 to 100 of such arrays can be fabricated on asingle substrate (such as glass). In such embodiment, after thesubstrate has the polynucleotides on its surface, the substrate may becut into substrate segments, each of which may carry one or two arrays.It will also be appreciated that there need not be any space separatingarrays from one another. Where a pattern of arrays is desired, any of avariety of geometries may be constructed, including for example,organized rows and columns of arrays (for example, a grid of arrays,across the substrate surface), a series of curvilinear rows across thesubstrate surface (for example, a series of concentric circles orsemi-circles of arrays), and the like.

The array substrate may take any of a variety of configurations rangingfrom simple to complex. Thus, the substrate could have generally planarform, as for example a slide or plate configuration, such as arectangular or square or disc. In many embodiments, the substrate willbe shaped generally as a rectangular solid, having a length in the rangeabout 4 mm to 300 mm, usually about 4 mm to 150 mm, more usually about 4mm to 125 mm; a width in the range about 4 mm to 300 mm, usually about 4mm to 120 mm and more usually about 4 mm to 80 mm; and a thickness inthe range about 0.01 mm to 5.0 mm, usually from about 0.1 mm to 2 mm andmore usually from about 0.2 to 1 mm. The substrate surface onto whichthe polynucleotides are bound may be smooth or substantially planar, orhave irregularities, such as depressions or elevations. Theconfiguration of the array may be selected according to manufacturing,handling, and use considerations.

In array fabrication, the quantities of polynucleotide available areusually very small and expensive. Additionally, sample quantitiesavailable for testing are usually also very small and it is thereforedesirable to simultaneously test the same sample against a large numberof different probes on an array. Therefore, one embodiment of theinvention provides for fabrication of arrays with large numbers of verysmall, closely spaced features. Arrays may be fabricated with featuresthat may have widths (that is, diameter, for a round spot) in the rangefrom a minimum of about 10 micrometers to a maximum of about 1.0 cm. Inembodiments where very small spot sizes or feature sizes are desired,material can be deposited according to the invention in small spotswhose width is in the range about 1.0 micrometer to 1.0 mm, usuallyabout 5.0 micrometers to 0.5 mm, and more usually about 10 micrometersto 200 micrometers. Interfeature areas will typically (but notessentially) be present that do not carry any polynucleotide. It will beappreciated, though, that the interfeature areas could be of varioussizes and configurations.

Suitable substrates may have a variety of forms and compositions and mayderive from naturally occurring materials, naturally occurring materialsthat have been synthetically modified, or synthetic materials. Examplesof suitable support materials include, but are not limited to,nitrocellulose, glasses, silicas, teflons, and metals (for example,gold, platinum, and the like). Suitable materials also include polymericmaterials, including plastics (for example, polytetrafluoroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike), polysaccharides such as agarose (e.g., that availablecommercially as Sepharose®, from Pharmacia) and dextran (e.g., thoseavailable commercially under the tradenames Sephadex® and Sephacyl®,also from Pharmacia), polyacrylamides, polystyrenes, polyvinyl alcohols,copolymers of hydroxyethyl methacrylate and methyl methacrylate, and thelike.

While the foregoing embodiments have been set forth in considerabledetail for the purpose of making a complete disclosure of the invention,it will be apparent to those of skill in the art that numerous changesmay be made in such details without departing from the spirit and theprinciples of the disclosure. Accordingly, the disclosure should belimited only by the following claims.

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties.

1. A method of forming polynucleotides, comprising: providing astructure X selected from:

where R1 is selected from substituted carbonyls, substituted silanes andfunctional groups, where the functional group is selected fromcarbonate, ester, amide, carbamate, silane, siloxane, orthoester,acetal, and ketal; where R2 is selected from benzyl, substituted benzyl,phenyl, substituted phenol, tertiary alkyl, and substituted tertiaryalkyl; where B comprises purine and pyrimidine bases and analogsthereof; wherein n is from 1 to 350; and wherein ● is a substrate; andoxidizing and deprotecting structure X simultaneously, wherein theaqueous buffer solution comprises an oxidant and a nucleophile at a pHof about 6 to
 10. 2. The method of claim 1, wherein the aqueous buffersolution is selected from hydrogen peroxide, peracids, performic acid,peracetic acid, perbenzoic acid, chloroperbenzoic acid, hydroperoxides,butylhydroperoxide, benzylhydroperoxide, phenylhydroperoxide, andcombinations thereof.
 3. The method of claim 1, wherein the aqueousbuffer solution comprises hydrogen peroxide.
 4. The method of claim 1,wherein R1 is selected from oxycarbonate, aryl ester, alkyl ester, alkylsilane, aryl silane, alkylsiloxane, arylsiloxane, alkylarylsilane, andalkylarylsiloxane.
 5. The method of claim 1, wherein R2 is selected fromalkylbenzyl, dialkylbenzyl, trialkylbenzyl, thioalkylbenzyl,phenylthiobenzyl, dithioalkylbenzyl, trithioalkylbenzyl,thioalkylhalobenzyl, alkyloxybenzyl, dialkyloxybenzyl, halobenzyl,dihalobenzyl, trihalobenzyl, and esterifiedsalicyl.
 6. The method ofclaim 1, wherein R1 is selected from oxycarbonate, aryl ester, alkylester, alkyl silane, aryl silane, alkylsiloxane, arylsiloxane,alkylarylsilane, and alkylarylsiloxane, and wherein R2 is selected fromalkylbenzyl, dialkylbenzyl, trialkylbenzyl, thioalkylbenzyl,phenylthiobenzyl, dithioalkylbenzyl, trithioalkylbenzyl,thioalkylhalobenzyl, alkyloxybenzyl, dialkyloxybenzyl, halobenzyl,dihalobenzyl, trihalobenzyl, and esterifiedsalicyl.
 7. The method ofclaim 1, wherein the pH of the aqueous buffer solution is about 6.5 to8.5.
 8. The method of claim 1, wherein the pH of the aqueous buffersolution is about 6.5 to
 8. 9. The method of claim 1, wherein the pH ofthe aqueous buffer solution is about 7 to
 8. 10. The method of claim 1,wherein oxidizing and deprotecting is an irreversible process.
 11. Themethod of claim 1, wherein oxidizing and deprotecting produces a productselected from:

wherein n is from 2 to 350.